The present invention relates to a method for preparing a vaccine for dental caries, a novel variant of Streptococcus mutans GS-5 as well as a vaccinal composition for dental caries which is used as nasal drops.
Streptococcus mutans group is the most crucial pathogenic factor in dental caries in human beings and animals. The S. mutans group is divided into 7 species depending on the characteristics of their DNA's and S. mutans of serotype c, which is most closely related to human dental caries, posesses, on its surface, substances having various antigenicities such as serotype-specific polysaccharide antigens, lipoteichoic acid and protein antigens.
One of these substances present on the bacterial cell surface, a protein antigen having a molecular weight of about 190,000 determined by SDS polyacrylamide gel electrophoresis, is a fine fibrous structure called fimbriae. This protein antigen is known variously as I/II, B, P1 and IF and it does not yet have a uniform name. Thus, this protein antigen is herein called PAc (protein antigen serotype c).
The protein antigen of S. mutans has thus come to atract much interest in connection with development of a vaccine for dental caries. For instance, Japanese Patent Un-examined Published Application (hereunder referred to as "J.P. KOKAI") No. Sho 56-501362 proposes the use of the antigen I/II posessing specific properties as a vaccine for dental caries and J.P. KOKAI No. Sho 58-164518 discloses a vaccine for preventing the decay of teeth. The vaccine comprises, as an antigen, the component of bacterial cells of microorganisms belonging to genus Streptococcus or an extract thereof.
However, even if S. mutans is cultured in 10 liters of a culture medium and PAc is extracted therefrom, only several mg of PAc can be recovered. Thus, this technique cannot be put to practical use. In addition, the purification process for removing impurities from the resulting culture medium requires a great deal of labor and time since the concentration of PAc in the culture medium is very low.
Under such circumstances, the inventors of this invention have tried to insert a PAc-expressing gene(pac gene) of S. mutans into a host such as E. coli (Escherichia coli) or S. milleri (a species of Streptococcus) to thus make the host cells produce rPAc. However, no increase in yield was achieved because the gene was integrated into the bacteria of different species and rPAc was not externally secreted. In this respect, the term "rPAc" herein means PAc obtained through gene recombination (recombinant PAc) and it is a protein having a structure substantially identical with that of PAc.
Moreover, the inventors linked the pac gene to a plasmid-shuttle vector which can proliferate in both E. coli and Streptococcus host cells and inserted it into a S. mutans GS-5 strain which cannot express PAc. The results are reported in Review of Nippon Dentistry Society, 1989, No. 555. However, this method suffers from various problems. For instance, after insertion of the pac gene into the cells of the GS-5 strain, it is sometimes observed that the plasmid carrying the gene is left out of the resulting recombinant bacterial cells and thus the variant stops the production of PAc and the products are insufficient in their uniformity.
On the other hand, there have been known a protective vaccine for dental caries which contains the foregoing protein antigen, i.e., PAc and the vaccine has been used through injection or oral administration.
However, if immunization is carried out by injection via skin, muscle or veins, local inflammation or even functional disorders may possibly be caused. In addition, since the injection is accompanied by pain, it is difficult to immunize an infant or a child through injection and patients are in general reluctant to have a vaccine injected at certain portions such as, for instance, near the parotid. Furthermore, it is difficult to increase the antibody titer in saliva by the administration through injection.
Moreover, if it is orally administered, it may be digested in the stomach. Also, oral administration is liable to causes immune tolerance and consequent loss of response to the corresponding antigen. Besides, a high antibody titer cannot be achieved by the oral immunization.